Journal: bioRxiv

Article Title: Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

doi: 10.1101/2020.05.20.103614

Figure Lengend Snippet: a. Schematic illustration of the TRAP-seq system. TLR, long terminal repeat; RRE, Rev Response Element; cPPT, central polypurine tract; U6, human U6 promoter; EFS-NS, short EFS promoter derived from EF1a. Figure not drown to scale. b. Schematic illustration of oligo pool synthesis, PCR amplification, gold-gate assembly, lentivirus packaging, and transduction of the 12K TRAP-seq library. c. Representative quantification of top 5 indel types for 3 TRAP sites. Dash line indicates the DSB site. Results for the 12,000 TRAP sites can be found at the CRISPR atlas database. d. Bar plots of SpCas9 editing efficiency of all TRAP sites measured by targeted amplicon sequencing. Results are shown for Dox-induced HEK293T-SpCas9 cells from 2, 8 and 10 days after transduction. Corresponding results for Dox-free HEK293T-SpCas9 are shown in Fig. S10. e. Bar plots of indel profiles (1-20 bp deletion, 1-10 bp insertion) for all TRAP sites introduced by SpCas9 in the Dox-induced HEK293T-SpCas9 cells from at 2, 8, and 10 days post transduction. Pie chat quantified the proportion of major indel groups: 1bp insertion (ins), 2bp insertion, 3-10 bp insertion, 1-12 bp deletion, 13-21bp deletion and 22-30 bp deletion. Other indels and wild-type reads are presented together as “others”. Corresponding results for Dox-free HEK293T-SpCas9 are shown in Fig. S11 and S12.

Article Snippet: Briefly, we replaced the SpCas9 open reading frame (ORF) in pLentiCRISPRv2-puro (Addgene plasmid # 98290) plasmid with an enhance green fluorescence protein (EGFP) ORF based on XbaI and BmHI digestion and T4 ligation.

Techniques: Derivative Assay, Amplification, Transduction, CRISPR, Sequencing

Journal: bioRxiv

Article Title: Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

doi: 10.1101/2020.05.20.103614

Figure Lengend Snippet: a. Box plot between GC content (with an interval of 10%) and SpCas9 gRNA efficiency measured in Dox-induced cells from Day 10. Results for other groups are shown in Fig. S14. b. Box plot between deltaG energy (with an interval of 2) and SpCas9 gRNA efficiency measured in Dox-induced cells from Day 10. Results for other groups are shown in Fig. S15. c. Comparison of SpCas9 efficiency between gRNAs harboring the GCC or TT motif in N17-N20 seed region and gRNAs without these two motifs. Data are shown for Dox-induced cells from Day 10. Results for other groups are shown in Fig. S16. “****”, p value less than 0.0001. d. Correlation between TRAP-seq indels and indels predicted by inDelphi from 11,910 sites. Data are shown for Dox-induced cells from Day 10. Results for other groups are shown in Fig. S17. e. Pie chart of the proportion of 1bp insertion among four bases (A, T, C, G) f. Correlation between the inserted 1 base and the nucleotide at N17 position. Data are shown for Dox-induced cells from Day 10. Results for other groups are shown in Fig. S18. g. Effects of N17N18 dinucleotide motifs on the indel frequency of 1bp insertion and deletions (1-30 bp). The gRNAs are divided into 16 groups based on the N17N18 motifs. For each gRNA, the total indel frequencies of 1-30bp deletions and 1bp insertion were analyzed. “n” indicates the number of gRNAs included for each group. h. Comparison of SpCas9 gRNA efficacy predictions in a regression schema for various datasets and prediction models. i. Top 20 features that weighted the most for the GNL machine learning model. Results are shown as the SHAP (SHapley Additive exPlanations) values. The 30mere comprises 4bp upstream, 20bp protospacer, 3 bp PAM, and 3 bp downstream sequences. Machine learning was based on gRNA efficiency data from Dox-induced cells at Day 10.

Article Snippet: Briefly, we replaced the SpCas9 open reading frame (ORF) in pLentiCRISPRv2-puro (Addgene plasmid # 98290) plasmid with an enhance green fluorescence protein (EGFP) ORF based on XbaI and BmHI digestion and T4 ligation.

Techniques: Comparison

Journal: bioRxiv

Article Title: Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

doi: 10.1101/2020.05.20.103614

Figure Lengend Snippet: a. Schematic illustration of the 6 stop-codon recoding schemes by CBE. Cas9 (D10A) is the Cas9 nickase used in the testing CBE editor. The stop-codon recoding scheme is draw based on sense and anti-sense strands. “ATG”, translation start site; “NGG”, PAM of SpCas9. b. Representation of top 5 cytosine base editing outcomes for three TRAP sites. Full CBE frequency results can be found in the CRISPR Atlas database. c. Quantification of overall CBE efficiency for all gRNAs in Dox-induced HEK293T-CBE cells at 11 days. Other groups are presented in Fig. S33. d. Quantification of overall percentage of C-to-T, C-to-G, C-to-A, and C-to-C (unedited) events across the 37bp region of all TRAP sites. Results are based on HEK293T-CBE cells from 2 days post transduction, and transduced HEK293T- CBE cells from 11 days cultured in Dox-free or Dox-addition medium. e. Representation of CBE-mediated recoding efficiency at one TRAP site measured by TRAP-seq. Complementary results referred to Fig. S35 and CRISPR atlas database. f. Comparison of C-to-T efficiency between sites within the 6 stop-codon recoding types. “n” indicates the number of sites within each group. “****”, p value less than 0.0001.

Article Snippet: Briefly, we replaced the SpCas9 open reading frame (ORF) in pLentiCRISPRv2-puro (Addgene plasmid # 98290) plasmid with an enhance green fluorescence protein (EGFP) ORF based on XbaI and BmHI digestion and T4 ligation.

Techniques: CRISPR, Transduction, Cell Culture, Comparison

Journal: bioRxiv

Article Title: Iterative engineering of a compact Cas9 ortholog for in vivo gene editing via single AAV delivery

doi: 10.64898/2026.01.12.698990

Figure Lengend Snippet: (A) Table of the Cas9 orthologs utilized in this work, showing their size (amino acid) and PAM sequence (5’). (B) Schematic size comparisons of the panel of Cas9 orthologs tested with SpCas9 and other compact CRISPR systems commonly used for in vivo genome editing. (C) Schematic outlining the experimental steps to evaluate Cas9 ortholog editing efficiency by tdTomato knock-out (KO), quantified by flow cytometry analysis. PiggyBac constructs encode for: ortholog-specific hU6-sgRNA cassettes and puromycin selection gene; doxycycline-inducible Cas9 orthologs and hygromycin selection gene. (D–E) Comparison of SpCas9 cleavage efficiency with Cco, Cme2, Gsp, Msc, Nsp, Sdo and Tmo Cas9 orthologs using three ortholog-specific spacer sequences targeting the tdTomato gene in mESC (D) and HEK293T cells (E). Editing efficiencies at individual target sites are shown in the left panels, while mean editing efficiencies for each Cas9 ortholog across the three sites are presented in the right panels. (F) Heatmaps showing editing efficiencies of Cme2 (left) and Msc (right) using twelve distinct sgRNAs, quantified by flow cytometry. Asterisks indicate P-values from unpaired t -test comparing three independent biological replicates to the respective controls. *P < 0.05. Error bars ± SD.

Article Snippet: SpCas9 coding region and sgRNA cassette were amplified from pX330-gRNA (Addgene #158973).

Techniques: Sequencing, CRISPR, In Vivo, Knock-Out, Flow Cytometry, Construct, Selection, Comparison